Hepatocyte transplantation (HT) could be of major value in the treatment of both inherited and acquired liver diseases. However, benefits of this procedure are currently limited by the number of hepatocytes that can be transplanted safely at a time. A method that would permit preferential proliferation of the engrafted cells over host hepatocytes would be of great value in repopulating the liver. In order to develop a clinically feasible preparative regimen for HT, we have explored preparative hepatic irradiation (HIR) for liver repopulation. Although preparative irradiation has been used routinely for bone marrow transplantation, we were the first to apply it to facilitate HT. Advances in conformal 3-dimensional and intensity-modulated radiation therapy permit the delivery higher radiation doses without liver failure. Our preliminary results demonstrate that preparative HIR and partial hepatectomy (PH), followed by HT results in the replacement of virtually all host hepatocytes by the transplanted non-irradiated hepatocytes in 12 weeks. Although PH provides a robust mitogenic stimulus, it is an invasive procedure, which is clinically applicable in limited situations, such as in patients requiring hepatic resection for liver cancer. To broaden the applicability of preparative HIR in the clinic, we need to (i) enhance the enqraftment and the rapidity and extent of hepatic repopuiation of the donor cells, (ii) design non-invasive alternatives to PH, (iii) reduce the required HiR dose or provide HIR to a portion of the liver for selective lobar repopulation, and (iv) develop a noninvasive method to monitor hepatic radiation injury and donor cell proliferation in the liver. We have designed our experiments to test the following Hypotheses - (1) HIR-induced oxidative damage to the sinusoidal endothelium of the liver should enhance engraftment of transplanted hepatocytes. (2) Since HT after PH+HIR normalizes hepatic radiation injury, we, hypothesize that hepatic metabolic state (assessed by the ATP/Pi index) would be restored in animals treated with PH+HIR+HT and 31P-MRS analyses of the liver would enable us to evaluate donor cell proliferation in the irradiated host liver. (3) Hepatotropic growth factors or methotrexate+HIR-induced necrosis should substitute for PH as a proliferative stimuli to transplanted hepatocytes. Since PH + HIR has become a standardized and reproducible preparative regimen in our laboratory, in Specific aim 1A, we will use the PH+HIR regimen as a model system to test variables aimed at improving donor cell engraftment, evaluation of the proliferative potential of various types of donor cells (e.g. large vs. small hepatocytes) and standardization of a noninvasive magnetic resonance-based assessment of metabolic recovery and donor cell proliferation. Once these variables are optimized in our PH+HIR regimen, we will use the optimized parameters in our HIR-based noninvasive regimens of HT. Specific aim IB will examine whether MRS can assess hepatic energy metabolism in irradiated animals and evaluate donor cell repopulation. In Specific aim 2A, we will examine whether hepatic growth factors, HGF and comitogens such as thyroid hormone can be used to substitute PH as a mitogenic stimulus for donor cells. In specific aim 2B, will test whether compensatory regenerative stimuli, provided by methotrexate + HIR-induced liver necrosis, can be used as a substitute to PH.